Abnormal splicing of ABCA1 pre-mRNA in Tangier disease due to a IVS2 +5G>C mutation in ABCA1 gene
2003; Elsevier BV; Volume: 44; Issue: 2 Linguagem: Inglês
10.1194/jlr.m200248-jlr200
ISSN1539-7262
AutoresSerena Altilia, Livia Pisciotta, Rita Garuti, Patrizia Tarugi, Alfredo Cantàfora, Laura Calabresi, Jacopo Tagliabue, Sergio Maccari, Franco Bernini, Ilaria Zanotti, Carlo Vergani, S Bertolini, S. Calandra,
Tópico(s)RNA and protein synthesis mechanisms
ResumoTwo point mutations of ABCA1 gene were found in a patient with Tangier disease (TD): i) G>C in intron 2 (IVS2 +5G>C) and ii) c.844 C>T in exon 9 (R282X). The IVS2 +5G>C mutation was also found in the brother of another deceased TD patient, but not in 78 controls and 33 subjects with low HDL. The IVS2 +5G>C mutation disrupts ABCA1 pre-mRNA splicing in fibroblasts, leading to three abnormal mRNAs: devoid of exon 2 (Ex2−/mRNA), exon 4 (Ex4−/mRNA), or both these exons (Ex2−/Ex4−/mRNA), each containing a translation initiation site. These mRNAs are expected either not to be translated or generate short peptides. To investigate the in vitro effect of IVS2 +5G>C mutation, we constructed two ABCA1 minigenes encompassing Ex1–Ex3 region, one with wild-type (WTgene) and the other with mutant (MTgene) intron 2. These minigenes were transfected into COS1 and NIH3T3, two cell lines with a different ABCA1 gene expression. In COS1 cells, WTgene pre-mRNA was spliced correctly, while the splicing of MTgene pre-mRNA resulted in Ex2−/mRNA. In NIH3T3, no splicing of MTgene pre-mRNA was observed, whereas WTgene pre-mRNA was spliced correctly.These results stress the complexity of ABCA1 pre-mRNA splicing in the presence of splice site mutations. Two point mutations of ABCA1 gene were found in a patient with Tangier disease (TD): i) G>C in intron 2 (IVS2 +5G>C) and ii) c.844 C>T in exon 9 (R282X). The IVS2 +5G>C mutation was also found in the brother of another deceased TD patient, but not in 78 controls and 33 subjects with low HDL. The IVS2 +5G>C mutation disrupts ABCA1 pre-mRNA splicing in fibroblasts, leading to three abnormal mRNAs: devoid of exon 2 (Ex2−/mRNA), exon 4 (Ex4−/mRNA), or both these exons (Ex2−/Ex4−/mRNA), each containing a translation initiation site. These mRNAs are expected either not to be translated or generate short peptides. To investigate the in vitro effect of IVS2 +5G>C mutation, we constructed two ABCA1 minigenes encompassing Ex1–Ex3 region, one with wild-type (WTgene) and the other with mutant (MTgene) intron 2. These minigenes were transfected into COS1 and NIH3T3, two cell lines with a different ABCA1 gene expression. In COS1 cells, WTgene pre-mRNA was spliced correctly, while the splicing of MTgene pre-mRNA resulted in Ex2−/mRNA. In NIH3T3, no splicing of MTgene pre-mRNA was observed, whereas WTgene pre-mRNA was spliced correctly. These results stress the complexity of ABCA1 pre-mRNA splicing in the presence of splice site mutations. Tangier disease (OMIM 600046) is a rare recessive disorder characterized by a severe deficiency or absence of HDL in plasma and an accumulation of cholesteryl esters in macrophages and other reticuloendothelial cells in many tissues (1Assmann G. von Eckardstein A. Brewer Jr, B.H. Familial Analphalipoproteinemia: Tangier Disease.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th edition. McGraw-Hill, New York2001: 2937-2960Google Scholar). Cholesteryl esters accumulation accounts for some of the clinical features of Tangier disease (TD), including orange-yellow tonsils, hepatosplenomegaly, peripheral nerve neuropathy, and corneal opacifications (1Assmann G. von Eckardstein A. Brewer Jr, B.H. Familial Analphalipoproteinemia: Tangier Disease.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th edition. McGraw-Hill, New York2001: 2937-2960Google Scholar, 2Oram J.F. Lawn R.M. ABCA1: the gatekeeper for eliminating excess of cholesterol.J. Lipid Res. 2001; 42: 1173-1179Google Scholar). The biochemical features of TD reflect a defect in the efflux of cholesterol and phospholipids from the cells due to a defective function of a membrane transporter designated ABCA1 (2Oram J.F. Lawn R.M. 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Zhang Z. Tsujii K.K. Matsuyama A. Ohama T. Matsuura F. Ishigami M. Sakai N. Hiraoka H. Hattori H. Wellington C. Yoshida Y. Misugi S. Hayden M.R. Egashira T. Yamashita S. Matsuzawa Y. Expression and functional analyses of novel mutations of ATP-binding cassette transporter-1 in Japanese patients with high-density lipoprotein deficiency.Biochem. Biophys. Res. Commun. 2002; 290: 713-721Google Scholar, 17Hong S.H. Rhyne J. Zeller K. Miller M. Novel ABCA1 compound variant associated with HDL cholesterol deficiency.Biochim. Biophys. Acta. 2002; 1587: 60-64Google Scholar). ABCA1 gene (GenBank no. AF275948) is located on chromosome 9 (9q31) and comprises 50 exons (18Santamarina-Fojo S. Peterson K. Knapper C. Qiu Y. Freeman L. Cheng J.F. Osorio J. Remaley A. Yang X.P. Haudenschild C. Prades C. Chimini G. Blackmon E. Francois T. Duverger N. Rubin E.M. Rosier M. Deneflè P. Fredickson D.S. Brewer Jr, B.H. Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATP-binding cassette A promoter.J. Clin. Invest. 2000; 97: 7987-7992Google Scholar). The open reading frame of the ABCA1 transcript is of 6,783 bp (18Santamarina-Fojo S. Peterson K. Knapper C. Qiu Y. Freeman L. Cheng J.F. Osorio J. Remaley A. Yang X.P. Haudenschild C. Prades C. Chimini G. Blackmon E. Francois T. Duverger N. Rubin E.M. Rosier M. Deneflè P. Fredickson D.S. Brewer Jr, B.H. Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATP-binding cassette A promoter.J. Clin. Invest. 2000; 97: 7987-7992Google Scholar, 19Pullinger C.R. Hakamata H. Duchateau N. Eng C. Aouizerat B.E. Cho M.H. Fielding C.F. Kane J.P. Analysis of hABC1 gene 5′ end: additional peptide sequence, promoter region, and four polymorphisms.Biochim. Biophys. Res. Commun. 2001; 271: 451-455Google Scholar), as opposed to the 6,603 described in an earlier report (20Langmann T. Klucken J. Reil M. Liebisch G. Luciani M-F. Chimini G. Kaminski W.E. Schmitz G. Molecular cloning of the human ATP-binding cassette transporter 1 (hABC1): evidence for sterol-dependent regulation in macrophages.Biochem. Biophys. Res. Commun. 1999; 257: 29-33Google Scholar). This transcript contains an in-frame ATG codon in exon 2, predicted to be the correct translation start site. This ATG codon is in a strong context, consistent with Kozak’s rules for initiation of translation (21Kozak M. Interpreting cDNA sequences: some insights from studies on translation.Mamm. Genome. 1996; 7: 563-574Google Scholar), in contrast with the ATG in exon 4 (a potential translation initiation site), which is in a weaker context with regard to these rules. The translation product of the full-length ABCA1 mRNA is a protein of 2,261 amino acids (18Santamarina-Fojo S. Peterson K. Knapper C. Qiu Y. Freeman L. Cheng J.F. Osorio J. Remaley A. Yang X.P. Haudenschild C. Prades C. Chimini G. Blackmon E. Francois T. Duverger N. Rubin E.M. Rosier M. Deneflè P. Fredickson D.S. Brewer Jr, B.H. Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATP-binding cassette A promoter.J. Clin. Invest. 2000; 97: 7987-7992Google Scholar, 19Pullinger C.R. Hakamata H. Duchateau N. Eng C. Aouizerat B.E. Cho M.H. Fielding C.F. Kane J.P. Analysis of hABC1 gene 5′ end: additional peptide sequence, promoter region, and four polymorphisms.Biochim. Biophys. Res. Commun. 2001; 271: 451-455Google Scholar), 60 amino acids more than originally reported (1Assmann G. von Eckardstein A. Brewer Jr, B.H. Familial Analphalipoproteinemia: Tangier Disease.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th edition. McGraw-Hill, New York2001: 2937-2960Google Scholar). This 60-amino-acid extension contains a potentially cleavable signal sequence, with cleavage predicted to occur between amino acids 45 and 46 (18Santamarina-Fojo S. Peterson K. Knapper C. Qiu Y. Freeman L. Cheng J.F. Osorio J. Remaley A. Yang X.P. Haudenschild C. Prades C. Chimini G. Blackmon E. Francois T. Duverger N. Rubin E.M. Rosier M. Deneflè P. Fredickson D.S. Brewer Jr, B.H. Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATP-binding cassette A promoter.J. Clin. Invest. 2000; 97: 7987-7992Google Scholar). The functional role of this extension has been recently elucidated in vitro using mutant ABCA1 cDNAs deleted at the 5′ end (22Fitzgerald M.L. Mendez A.J. Moore K.J. Andersson L.P. Panjecton H.A. Freeman M.W. ATP-binding cassette transporter A1 contains an NH2-terminal signal anchor sequence that translocates the protein’s first hydrophilic domain to the exoplasmic space.J. Biol. Chem. 2001; 276: 15137-15145Google Scholar). These studies showed that: a) the 60-amino-acid extension is required for the stable protein expression of transporter constructs containing any downstream transmembrane domains, and b) the putative signal sequence contained in this extension is not cleaved from the protein. It apparently serves as an anchor sequence that positions the N-terminus of ABCA1 in a type II orientation, leading to the extracellular presentation of a 600-amino-acid loop (22Fitzgerald M.L. Mendez A.J. Moore K.J. Andersson L.P. Panjecton H.A. Freeman M.W. ATP-binding cassette transporter A1 contains an NH2-terminal signal anchor sequence that translocates the protein’s first hydrophilic domain to the exoplasmic space.J. Biol. Chem. 2001; 276: 15137-15145Google Scholar). These in vitro observations suggest that mutations in ABCA1 gene, which eliminate (or disrupt) the 60 amino acid extension, are likely to prevent the synthesis of ABCA1 protein, leading to a complete deficiency of this transporter in the cells. There are some indications that under normal physiological conditions, ABCA1 pre-mRNA is subject to alternative splicing. Costet et al. (23Costet P. Luo Y. Wang N. Tall A.R. Sterol-dependent transactivation of the ABCA1 promoter by the liver X receptor/retinoid X receptor.J. Biol. Chem. 2000; 276: 28240-28245Google Scholar) reported three alternative transcripts of ABCA1 gene in HepG2 cells involving the exon 1–exon 5 region. We demonstrated the presence of an alternatively spliced ABCA1 mRNA lacking exon 4 in several human cell types, including normal skin fibroblasts (24Bellincampi L. Simone M.L. Motti C. Cortese C. Bernardini S. Bertolini S. Calandra S. Identification of an alternative transcript of ABCA1 gene in different human cell types.Biochim. Biophys. Res. Commun. 2001; 283: 590-597Google Scholar). A recent study conducted in human ABCA1 transgenic mice showed the presence of a transcript containing an alternative exon 1, followed by correctly spliced exons 2–4 (25Cavelier L.B. Qiu Y. Bielicki J.K. Afzal V. Cheng J-F. Rubin E.M. Regulation and activity of the human ABCA1 gene in transgenic mice.J. Biol. Chem. 2001; 276: 18046-18051Google Scholar). This newly discovered exon, named exon 1A, is located 2,210 bp upstream from exon 2. In the present study, we report the effects of a point mutation in intron 2 of ABCA1 gene found in a patient with Tangier disease. This mutation disrupts the normal splicing, causing the formation of several abnormal ABCA1 mRNAs in cultured skin fibroblasts. One of these mRNAs does not contain exon 2; therefore, it is predicted to encode an ABCA1 protein devoid of the 60-amino-acid extension involved in anchoring the transporter to the plasma membrane. Another of these abnormal mRNAs is devoid of both exon 2 and exon 4, which contain the two translation initiation sites. The proband was a 66-year-old female (I.1 in Fig. 1)with severe HDL deficiency (Table 1). She had had tonsillectomy at the age of 8. Since the age of 20, she had secondary amenorrhea caused by primary hypogonadism with ovary and uterine hypoplasia. She was a heavy smoker (40 cigarettes/day). At the age of 57, she was found to have a thoracoabdominal aortic aneurysm with aortic valve insufficiency. At 62, the diameter of the descending thoracic aorta was 6.5 cm. The patient was not surgically treated because of the exceptionally high operative risk. At 67, the thoracoabdominal aortic diameter was 9.0 cm and the patient died from aortic dissection and rupture. The proband's apparently healthy brother (I.2) had hypoalphalipoproteinemia. The proband's sister (I.3) had normal lipid values (Table 1).TABLE 1Clinical characteristics of the subjects investigatedFamily 1Family 2Subject (sex)I.1 (F)I.2 (M)I.3 (F)I.1 (M)I.3 (M)II.1 (F)II.2 (F)Age (years)66655762945754Cholesterol (mmol/l)2.822.795.942.02.795.925.22HDL-cholesterol (mmol/l)0.080.521.470.050.441.061.11Triglycerides (mmol/l)2.181.482.001.61.811.240.87ApoA-I (mg/dl)<1062151 C transversion (see Results) introduces a new StyI restriction site in the ABCA1 gene. To screen for the presence of this mutation, genomic DNA was amplified using the following primers: 5′-GCTGGATTAGCAGTCCTCATTG-3′ (forward primer in exon 2) and 5′-CCCCAACTCAAAACCACAAAG-3′ (reverse primer in intron 2). DNA was amplified in a 50 μl volume containing 0.2 mM of each dNTP, 20 pmol of each primer, 1 U Taq DNA polymerase in 1× PCR buffer, and 2 mM MgCl2. The conditions were 94°C for 3 min, followed by 30 cycles of 94°C
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